Regulation of PDGF-stimulated SHIP2 tyrosine phosphorylation and association with Shc in 3T3-L1 preadipocytes

Ottawa Health Research Institute, Department of Medicine, University of Ottawa, Ottawa, Ontario, Canada.
Journal of Cellular Physiology (Impact Factor: 3.84). 06/2007; 211(3):598-607. DOI: 10.1002/jcp.20965
Source: PubMed


In 3T3-L1 and human preadipocytes, insulin results in the isolated rise in phosphatidylinositol (PI)-3,4,5-P3, whereas PDGF produces PI(3,4)P2 in addition to PI(3,4,5)P3. SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) converts PI(3,4,5)P3 into PI(3,4)P2. PDGF, but not insulin, stimulates SHIP2 tyrosine phosphorylation and its association with Shc in human and 3T3-L1 preadipocytes. We now demonstrate that SHIP2 tyrosine phosphorylation and association with Shc in PDGF-treated 3T3-L1 preadipocytes was reduced by bisindolylmaleimide I (BisI), an inhibitor of conventional/novel protein kinase C (PKC). However, the production of PI(3,4)P2 and PI(3,4,5)P3 by PDGF was unaffected by BisI. Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) was not sufficient to induce SHIP2 tyrosine phosphorylation. Furthermore, we identified threonine 958 (T958) as a novel PDGF-responsive SHIP2 phosphorylation site. Mutation of T958 to alanine reduced PDGF-stimulated SHIP2 tyrosine phosphorylation and association with Shc, but did not alter its anti-proliferative effect on preadipocytes. This study demonstrates that SHIP2 tyrosine phosphorylation and Shc association can be regulated by serine/threonine signaling pathways, either indirectly (via PKC), or directly (via T958). Interestingly, the anti-proliferative effect of SHIP2 T958A, as well as another SHIP2 mutant (Y986F, Y987F) that also displays defective tyrosine phosphorylation and Shc association, does not depend on these molecular events.

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    ABSTRACT: Class I phosphoinositide (PI) 3-kinases exert profound effects on cell growth, division, motility and metabolism via their primary lipid product phosphatidylinositol 3,4,5- trisphosphate (PtdIns(3,4,5)P3) and a metabolite of this, phosphatidylinositol 3,4- bisphosphate (PtdIns(3,4)P2). Many effector proteins for PtdIns(3,4,5)P3 are well recognised but by contrast, few molecular targets for PtdIns(3,4)P2 have been identified. This study describes a screen to identify PI 3-kinase-responsive proteins that is selective particularly for these. The approach features a unique three-tier affinity approach and incorporates a primary recruitment of target proteins to membranes of intact cells, selectively enriched in PtdIns(3,4)P2. In addition, this screen utilises stable isotope labelling with amino acids in cell culture (SILAC) to differentially label cells stimulated in the absence and presence of the PI 3-kinase inhibitor wortmannin. The integration of these techniques provides a ratio-metric readout, allowing authentically 3- phosphoinositide (3-PI) responsive components to be distinguished from the co-purifying background proteins. The identification of tandem pleckstrin homology domain containing protein-1 (TAPP-1) and protein kinase B (PKB) among a multitude of proteins expressing known lipid binding domains (LBDs) demonstrates the utility of this strategy. Analysis of other similarly, isotopically enriched candidate 3-PI interacting proteins yielded two novel lipid binding proteins, PARIS-1 (prostate antigen recognised and identified by SEREX 1) and IQGAP1 (IQ motif containing GAP1). The concentration dependent interaction of PARIS-1 and IQGAP1 with PtdIns(3,4,5)P3 was confirmed by an in vitro, SPR based assay. Intriguingly, IQGAP1, a potential tumour promoter, lacks a currently established LBD and may therefore exemplify an entirely novel 3-PI selective binding domain.
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    ABSTRACT: SHIP2 (SH2-containing inositol polyphosphate 5-phosphatase 2) is a phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) 5-phosphatase containing various motifs susceptible to mediate protein-protein interaction. In cell models, SHIP2 negatively regulates insulin signalling through its catalytic PtdIns(3,4,5)P(3) 5-phosphatase activity. We have previously reported that SHIP2 interacts with the c-Cbl associated protein (CAP) and c-Cbl, proteins implicated in the insulin cellular response regulating the small G protein TC10. The first steps of the TC10 pathway are the recruitment and tyrosine phosphorylation by the insulin receptor of the adaptor protein with Pleckstrin Homology and Src Homology 2 domains (APS). Herein, we show that SHIP2 can directly interact with APS in 3T3-L1 adipocytes and in transfected CHO-IR cells (Chinese hamster ovary cells stably transfected with the insulin receptor). Upon insulin stimulation, APS and SHIP2 are recruited to cell membranes as seen by immunofluorescence studies, which is consistent with their interaction. We also observed that SHIP2 negatively regulates APS insulin-induced tyrosine phosphorylation and consequently inhibits APS association with c-Cbl. APS, which specifically interacts with SHIP2, but not PTEN, in turn, increases the PtdIns(3,4,5)P(3) 5-phosphatase activity of SHIP2 in an inositol phosphatase assay. Co-transfection of SHIP2 and APS in CHO-IR cells further increases the inhibitory effect of SHIP2 on Akt insulin-induced phosphorylation. Therefore, the interaction between APS and SHIP2 provides to both proteins potential negative regulatory mechanisms to act on the insulin cascade.
    Journal of Cellular Physiology 01/2008; 214(1):260-72. DOI:10.1002/jcp.21193 · 3.84 Impact Factor
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    ABSTRACT: Inadequate proliferation and/or differentiation of preadipocytes may lead to adipose tissue dysfunction characterized by hypertrophied, insulin-resistant adipocytes. Platelet-derived growth factor (PDGF) may alter adipose tissue function by promoting proliferation of preadipocytes. Two principal signaling pathways that regulate proliferation are PI3K/PI(3,4,5)P3/Akt and Shc/Ras/ERK1/2. SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) dephosphorylates PI(3,4,5)P3, and also binds to Shc. Our goal was to determine how SHIP2 affects these PDGF signaling routes. To assess the role of the 5-phosphatase domain, we expressed wild-type or catalytically inactive dominant-negative SHIP2 (P686A-D690A-R691A; PDR/AAA) in 3T3-L1 preadipocytes. Surprisingly, SHIP2 PDR/AAA inhibited proliferation more potently than wild-type SHIP2. After three days of proliferation, phospho-Akt, phospho-ERK1/2, and PDGF receptor (PDGFR) levels were reduced in PDR/AAA-expressing preadipocytes. SHIP2 PDR/AAA interference with PDGFR signaling was demonstrated using imatinib, an inhibitor of PDGFR tyrosine kinase. The anti-proliferative effect of imatinib observed in control preadipocytes was not significant in SHIP2 PDR/AAA-expressing preadipocytes, indicating a pre-existing impairment of PDGFR-dependent mitogenesis in these cells. The inhibition of PDGF-activated mitogenic pathways by SHIP2 PDR/AAA was consistent with a decrease in PDGFR phosphorylation caused by a drop in receptor levels in SHIP2 PDR/AAA-expressing cells. SHIP2 PDR/AAA promoted ubiquitination of the PDGFR and its degradation via the lysosomal pathway independently of the association between the E3 ubiquitin ligase c-Cbl and PDGFR. Overall, our findings indicate that SHIP2 PDR/AAA reduces preadipocyte proliferation by attenuating PDGFR signaling.
    Journal of Cellular Physiology 01/2009; 218(1):228-36. DOI:10.1002/jcp.21595 · 3.84 Impact Factor
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